Certain bacterial pathogens have been found to possess multiple redundant iron acquisition systems that may be activated in response to iron limiting growth conditions in the host. Bordetella pertussis and B. bronchiseptica are Gram-negative bacterial pathogens that colonize the respiratory mucosal epithelia of humans and nonhuman mammals. These organisms can accumulate iron supplied by their native macrocyclic dihydroxamate siderophore, alcaligin, as well as siderophores produced by other microbial species. Additionally, in vitro growth stimulation by the host iron-containing compounds heme, hemoglobin, lactoferrin, and transferrin has been reported for these Bordetella spp. From this range of potential iron sources available to support Bordetella growth in the host, it is unknown which are the effective sources during the stages of infection. However, conservation of the alcaligin siderophore system from the evolutionarily-related Alcaligenes genus to the strictly human pathogen B. pertussis suggests that this means of iron retrieval is important for survival in the host. The goal of this research project is to identify and characterize the mechanisms regulating the in vitro and in vivo expression of iron acquisition systems of B. pertussis and to evaluate the importance of the native siderophore alcaligin in virulence. The specific aims of the proposal are to 1) elucidate the mechanism of gene regulation by alcaligin, 2) characterize the function of AlcR, a putative transcriptional regulator, and 3) evaluate the importance of AlcR and alcaligin for virulence. The factors controlling the expression of the alcaligin biosynthesis operon will be characterized. The mechanisms by which alcaligin and the putative transcriptional regulator AlcR govern the expression of alc genes and other known iron acquisition systems will be examined in detail and the established role of the Fur repressor evaluated in the context of that model system. Virulence of alcaligin biosynthesis and regulatory mutants will be evaluated in appropriate models of infection. These studies will provide the framework for analysis of the coordinated bacterial response to iron starvation in the host environment and will lead to greater understanding of B. pertussis physiology and pathogenesis.